Fig 2: Identification of mutations in GON7 and YRDC in patients with Galloway-Mowat syndrome. a, c Pedigrees of families with mutations in GON7 (a) and in YRDC (c). Affected individuals are in black. b, d Organization of exons of human GON7 and YRDC cDNAs. Positions of start and stop codons are indicated. Arrows indicate positions of the identified mutations. Lower panels show the sequencing traces for affected individuals with identified mutated nucleotide indicated with a red square (Hom: homozygous; het, heterozygous). e Representation of a 3D model of human YRDC, bound to the reaction product threonylcarbamoyl-adenylate, in sticks. The model was constructed using the crystal structure of Sulfolobus tokodaii Sua5 (PDB code 4E1B). The side chains of the three mutated residues are in black sticks. The green sphere represents an Mg2+ ion

Fig 3: Proliferation, apoptosis, and protein synthesis defects upon GON7 and YRDC knockdown. Transient knockdown (KD) of GON7, LAGE3, YRDC, and OSGEP was performed by lentiviral transduction of shRNA in immortalized human podocyte cell lines with a scrambled (non-targeting) shRNA as control. a Cell proliferation was assessed using a colorimetric MTT assay over 7 days, measuring absorbance at 490 nm at days 1, 2, 3, 4, and 7 (mean ± s.e.m. of n = 5 experiments, with each experiment performed in triplicate; two-way ANOVA (p < 0.0001), Dunnett’s multiple comparisons test, n.s. = 0.2031, ***p < 0.0007, ****p < 0.0001). b Cell apoptosis was evaluated by quantification of caspase 3/7 activation on the basis of fluorescence intensity (530/405 nm). Absolute values were normalized to DAPI fluorescence intensity as an internal control and compared to non-targeting shRNA-treated control cells (scrambled) (mean ± s.e.m. of n = 3 experiments with each experiment performed in triplicate; one-way ANOVA (F (4,10) = 21.42, p < 0.0001), Dunnett’s multiple comparisons test, n.s. = 0.0556, **p = 0.0012, ****p < 0.0001). c Protein biosynthesis rates were assessed on the basis of incorporation of HPG, an alkyne-containing methionine analog. After 2 h, alkyne-containing proteins were quantified on the basis of fluorescence intensity (485/535 nm). Absolute values were normalized to DAPI fluorescence intensity as an internal control and compared to control cells (mean ± s.e.m. of n = 3 experiments, with each experiment performed in triplicate, one-way ANOVA (F (4,10) = 16.36, p= 0.0002), Dunnett’s multiple comparisons test, **p = 0.0035, ****p < 0.0003). d Relative expression of GON7, YRDC, LAGE3, and OSGEP transcripts were normalized to that of HPRT in KD podocytes compared to non-targeting shRNA control treated cells (mean ± s.e.m. of n = 5 experiments, with each experiment being performed in triplicate; two-tailed Mann–Whitney test, **p < 0.05). Source data are provided as a Source Data file

Fig 4: Effects of YRDC and GON7 mutations on t6A biosynthesis. a Evaluation of fitness of ?sua5 yeast strains expressing human YRDC variants (spots are from 10-fold serial dilutions of cell suspensions at OD600nm = 0.5, and three independent clones were evaluated) and b western blot analysis on total protein extracts from ?sua5 yeast cells expressing human YRDC variants using anti-hYRDC antibody. c, d Mass spectrometry (LC-MS/MS) quantification of t6A modification in total tRNAs extracted from ?sua5 yeast cells expressing human YRDC variants (c) (mean ± s.e.m. of two independent LC-MS/MS experiments (technical replicates), each measuring samples from three independent yeast transformants; one-way ANOVA (F (5,28) = 269.4, p < 0.0001), Dunnett’s multiple comparisons test, ***p = 0.0008, ****p < 0.0001) and from cultured primary skin fibroblasts from controls (two unaffected individuals) and affected individuals with either the p.Tyr7* GON7 mutation (two individuals), or the YRDC mutations (three individuals) or with the p.Arg352Gln OSGEP mutation in the homozygous state (individual «CP» described in Braun et al.13) (d) (mean ± s.e.m. of two independent LC-MS/MS experiments (technical replicates), each measuring samples from three independent cell culture experiments; one-way ANOVA (F (3,44) = 6.446, p < 0.001), Dunnett’s multiple comparisons test, n.s. = 0.4894, *p = 0.0169, ***p = 0.008). Source data are provided as a Source Data file